本文選題：雄激素受體 + 拮抗劑��； 參考：《山東大學(xué)》2016年博士論文

【摘要】：研究目的：前列腺癌(Prostate cancer, PCa)是男性泌尿生殖系統(tǒng)中最常見的惡性腫瘤,嚴(yán)重威脅著男性健康。我國(guó)是前列腺癌發(fā)病率和死亡率均較低的國(guó)家之一。但近年來,隨著我國(guó)人民生活水平的提高、飲食結(jié)構(gòu)的改變及醫(yī)療診斷水平的提高,其發(fā)病率呈現(xiàn)快速增長(zhǎng)趨勢(shì)。早期局限性PCa可以通過手術(shù)治療和放療完全治愈。對(duì)于非局限性、不能手術(shù)的PCa患者,根據(jù)PCa生長(zhǎng)依賴于雄激素和雄激素受體(Androgen receptor,AR)信號(hào)通路的特性,通常采用雄激素去勢(shì)聯(lián)合應(yīng)用雄激素受體拮抗劑的雄激素阻斷療法(Androgen deprivation therapy, ADT)進(jìn)行治療。起初,ADT對(duì)大多數(shù)患者有效,但是經(jīng)過一段時(shí)間的治療后,患者會(huì)復(fù)發(fā)并進(jìn)展為致命的去勢(shì)抵抗性前列腺癌(Castration resisitant PCa, CRPC)。CRPC對(duì)抗雄激素藥物治療耐受,這種現(xiàn)象在臨床上稱為“抗雄激素抵抗”。CRPC目前尚無有效的治療方法,其發(fā)生進(jìn)展機(jī)制也尚未闡明,但目前普遍認(rèn)為其與AR信號(hào)通路的重新激活密切相關(guān),因此,AR是治療前列腺癌的關(guān)鍵靶標(biāo)。本研究論文以前列腺癌發(fā)生發(fā)展過程起關(guān)鍵作用的分子AR為靶點(diǎn),基于AR配體結(jié)構(gòu)域(Ligand Binding Domain, LBD)的晶體結(jié)構(gòu)以及現(xiàn)有AR拮抗劑與ARLBD的作用模式,綜合運(yùn)用藥物化學(xué)、化學(xué)生物學(xué)和計(jì)算機(jī)化學(xué)等藥物設(shè)計(jì)和發(fā)現(xiàn)策略,設(shè)計(jì)合成結(jié)構(gòu)全新的AR拮抗劑做為抗前列腺癌候選藥物。研究方法：首先,我們根據(jù)Me-too藥物的設(shè)計(jì)原則,對(duì)用于CRPC治療的第二代AR拮抗劑恩雜魯胺進(jìn)行微小的結(jié)構(gòu)改造,設(shè)計(jì)合成一系列乙內(nèi)酰硫脲類衍生物。該類化合物的合成以2-取代對(duì)硝基苯甲酸和取代苯胺為原料,通過縮合反應(yīng)、還原反應(yīng)、strecker反應(yīng)、及硫代乙內(nèi)酰脲成環(huán)反應(yīng)制備。該系列化合物的生物活性評(píng)價(jià)通過實(shí)時(shí)熒光定量PCR法、熒光素酶報(bào)告基因法和MTT法分別對(duì)其前列腺特異性抗原(Prostate specific antigen, PSA)表達(dá)抑制活性、AR轉(zhuǎn)錄抑制活性和抗增殖活性進(jìn)行測(cè)定。由于目前臨床上應(yīng)用的的AR拮抗劑都包含相同的結(jié)構(gòu)片段,我們推測(cè)這可能與耐藥性的產(chǎn)生有關(guān)。為了尋找具有全新結(jié)構(gòu)骨架的小分子AR拮抗劑,我們用計(jì)算機(jī)輔助藥物設(shè)計(jì)軟件SYBLE X1.1中的藥效團(tuán)構(gòu)建模塊GALAHAD以五個(gè)AR拮抗劑為模板構(gòu)建3D藥效團(tuán),并用UNITY模塊產(chǎn)生藥效團(tuán)模型3D提問式對(duì)商業(yè)化合物數(shù)據(jù)庫搜索,尋找符合藥效團(tuán)模型特征的化合物,用Surflex dock模塊將得到的化合物與野生型AR和突變型AR對(duì)接,將與兩個(gè)受體對(duì)接打分值都大于7的化合物進(jìn)行結(jié)合模式分析,挑選出結(jié)合模式較好且能夠從商業(yè)途徑獲得的化合物。然后通過熒光偏振法、實(shí)時(shí)熒光定量PCR法和MTT法對(duì)虛擬篩選得到的化合物的AR親和力、PSA抑制活性和前列腺癌細(xì)胞生長(zhǎng)抑制活性進(jìn)行評(píng)價(jià)。虛擬篩選過程中發(fā)現(xiàn)的化合物T3的PSA抑制活性較低,為了提高T3的AR轉(zhuǎn)錄抑制活性和抗增殖活性,我們對(duì)化合物T3進(jìn)行了結(jié)構(gòu)改造,通過對(duì)中間連接鏈和兩端芳環(huán)不同取代基的考察,設(shè)計(jì)合成一系列吡唑衍生物進(jìn)行構(gòu)效關(guān)系研究。該類化合物的合成以取代苯乙酮為原料,經(jīng)克萊森縮合反應(yīng)、吡唑成環(huán)反應(yīng)、N-烷基化反應(yīng)、水解反應(yīng)、傅-克反應(yīng)及縮合反應(yīng)制備。通過實(shí)時(shí)熒光定量PCR法和MTT法對(duì)該類化合物的PSA表達(dá)抑制活性和抗增殖活性進(jìn)行測(cè)定。本論文合成的化合物結(jié)構(gòu)通過核磁共振氫譜(1H-Nuclear Magnetic Resonance, 1H-NMR)、核磁共振碳譜(13C-Nuclear Magnetic Resonance,13C-NMR)和高分辨質(zhì)譜(High Resolution Mass Spectra, HRMS)等方法進(jìn)行確證,并用高效液相色譜法(Hign Performance Liquid Chromatography, HPLC)進(jìn)行了純度測(cè)定,RY-1型顯微熔點(diǎn)測(cè)定儀測(cè)定了目標(biāo)化合物的熔點(diǎn)。經(jīng)查閱文獻(xiàn)證實(shí),除個(gè)別化合物外所合成的目標(biāo)化合物均為新化合物,未見文獻(xiàn)報(bào)道。研究結(jié)果：通過研究乙內(nèi)酰硫脲類衍生物對(duì)野生型和T877A突變型AR的拮抗激動(dòng)活性和抗增殖活性,發(fā)現(xiàn)大部分的化合物與恩雜魯胺一樣,能夠選擇性抑制表達(dá)AR的LNCaP細(xì)胞的增殖,而對(duì)雄激素非依賴的PC-3細(xì)胞無作用。其中7t是活性最好的化合物,其對(duì)前列腺癌細(xì)胞LNCaP的生長(zhǎng)抑制活性(Gl50=1.78μM)較恩雜魯胺(GI50＝23.58μM)提高了15倍�；衔�7t表現(xiàn)出與恩雜魯胺相似的選擇性抑制LNCaP細(xì)胞生長(zhǎng)的特點(diǎn),然而其對(duì)AR的轉(zhuǎn)錄抑制活性較恩雜魯胺有所下降,對(duì)去勢(shì)條件下LNCaP細(xì)胞生長(zhǎng)抑制活性較恩雜魯胺高,說明化合物7t的作用機(jī)制可能與恩雜魯胺不同�；衔�7i和7j對(duì)AR轉(zhuǎn)錄抑制活性和LNCaP細(xì)胞生長(zhǎng)抑制活性較恩雜魯胺更高,它們同時(shí)對(duì)不表達(dá)AR的前列腺癌細(xì)胞PC-3也有很強(qiáng)的的抑制活性,說明化合物7i和7j可能通過AR依賴和AR非依賴雙重途徑發(fā)揮作用�；衔�7j和7t可能在CRPC的治療中較恩雜魯胺有優(yōu)勢(shì),其作用機(jī)制和成藥性值得進(jìn)一步研究。虛擬篩選過程中構(gòu)建的藥效團(tuán)模型含有三個(gè)疏水性中心和三個(gè)氫鍵受體六個(gè)特征元素,與之前文獻(xiàn)報(bào)道的藥效團(tuán)特征部分相符。通過對(duì)虛擬篩選得到的9個(gè)具有不同結(jié)構(gòu)骨架的小分子的活性測(cè)試發(fā)現(xiàn),9個(gè)小分子化合物均能與二氫睪酮(Dihydrotestosterone, DHT)競(jìng)爭(zhēng)性結(jié)合AR、抑制AR的轉(zhuǎn)錄活性,證明了我們基于藥效團(tuán)和分子對(duì)接的虛擬篩選方案的可行性�；衔颰3、T4、T6和T8與MDV3100相似,均能夠選擇性抑制雄激素依賴的前列腺癌細(xì)胞LNCaP的生長(zhǎng),而對(duì)雄激素非依賴的前列腺癌細(xì)胞PC-3無明顯影響。其中,化合物T3能夠與DHT競(jìng)爭(zhēng)性結(jié)合AR,顯著抑制AR的轉(zhuǎn)錄活性,并且對(duì)雄激素依賴的前列腺癌細(xì)胞LNCaP的生長(zhǎng)抑制活性(GI50=16.52μM)優(yōu)于MDV3100�；衔颰3可以作為研究AR拮抗劑的先導(dǎo)化合物,進(jìn)行進(jìn)一步結(jié)構(gòu)修飾。通過對(duì)先導(dǎo)化合物T3的結(jié)構(gòu)修飾,我們?cè)O(shè)計(jì)合成了吡唑-5-酮類衍生物和吡唑-5-甲酰胺類衍生物。我們對(duì)吡唑-5-甲酰胺類衍生物兩端苯環(huán)和吡唑1位N上的取代基進(jìn)行了考察,得到一系列吡唑-5-甲酰胺類衍生物。通過測(cè)試該類化合物對(duì)AR的轉(zhuǎn)錄抑制活性,總結(jié)吡唑-5-甲酰胺類衍生物的構(gòu)效關(guān)系為：1)R1位上的取代基CF3,FC1、Br和CH3；2)R2位上甲基或乙基取代對(duì)PSA的抑制活性無明顯影響；3)R4位單取代優(yōu)于R3或R5,R4位上單取代溴、氯、三氟甲基、氰基和硝基優(yōu)于乙炔基、氟和氫；當(dāng)R4位為三氟甲基時(shí),在R5位引入鹵素能夠提高化合物對(duì)PSA的抑制活性。該類化合物中活性最好的兩個(gè)化合物是H25和H43,與先導(dǎo)化合物T3相比：1)對(duì)PSA的抑制活性有所提高：H25和H43在10μM濃度下對(duì)PSA的抑制活性分別為100.00%和87.90%,而先導(dǎo)化合物T3和陽性對(duì)照藥恩雜魯胺分別為60.71%和96.79%；2)對(duì)LNCaP細(xì)胞的生長(zhǎng)抑制活性提高了3-4倍：H25和H43的GIso分別為7.73和3.54μM,而先導(dǎo)化合物T3和比卡魯胺的G150分別為16.52和23.11μM;3)能夠抑制雄激素非依賴性前列腺癌細(xì)胞PC-3的生長(zhǎng)。以上結(jié)果提示這些化合物可能通過AR依賴性和AR非依賴性雙重途徑發(fā)揮作用,具體作用機(jī)制有待進(jìn)一步闡明。結(jié)論及意義：本研究論文以AR為靶點(diǎn),基于ARLBD的晶體結(jié)構(gòu)以及現(xiàn)有AR拮抗劑與ARLBD的相互作用模式,以恩雜魯胺為先導(dǎo)設(shè)計(jì)合成了一系列乙內(nèi)酰硫脲衍生物；同時(shí)運(yùn)用基于藥效團(tuán)和分子對(duì)接的虛擬篩選策略發(fā)現(xiàn)了骨架結(jié)構(gòu)全新的先導(dǎo)化合物T3,并對(duì)T3進(jìn)行結(jié)構(gòu)修飾設(shè)計(jì)合成了吡唑-5-酮類衍生物和吡唑-5-甲酰胺類衍生物,總結(jié)了吡唑-5-甲酰胺類衍生物的構(gòu)效關(guān)系。通過初步的體外活性測(cè)試,我們發(fā)現(xiàn)乙內(nèi)酰脲類衍生物7j和7t可能在CRPC的治療中較恩雜魯胺有優(yōu)勢(shì),其作用機(jī)制和成藥性有待進(jìn)一步研究。以上結(jié)果為我們?cè)O(shè)計(jì)合成新的AR拮抗劑提供了依據(jù)。
[Abstract]:Research purposes : Prostate cancer ( PCa ) is one of the most common malignant tumors in the male urinary system , which is a serious threat to male health . In recent years , with the improvement of the living standard of our people , the change of dietary structure and the improvement of medical diagnosis level , the incidence of PCa can be completely cured by surgical treatment and radiotherapy . In order to find a new drug for the treatment of prostate cancer , a new type of AR antagonist was designed and synthesized by means of real - time fluorescence quantitative PCR , luciferase reporter gene method and MTT method . The inhibitory activity of compound 7i and 7j on the growth inhibitory activity ( G50 = 23.58 渭M ) of wild type and T877A was studied . A series of pyrazole - 5 - carboxamide derivatives are obtained . Through testing the transcriptional inhibitory activity of the compounds on the AR , the structure - effect relationship of the pyrazole - 5 - carboxamide derivatives is summarized as follows : 1 ) the substituents CF3 , FC1 , Br and CH3 on the R1 position ;
2 ) the inhibition activity of methyl or ethyl substitution on the R2 position has no obvious effect on the inhibition activity of the PSA ;
3 ) R4 position mono - substituted is better than R3 or R5 , and the mono - substituted bromine , chlorine , trifluoromethyl , cyano and nitro groups on the R4 position are better than acetylene group , fluorine and hydrogen ;
When R4 is trifluoromethyl , halogen can be introduced at the R5 position to increase the inhibitory activity of the compound on PSA . The best two compounds of these compounds are H25 and H43 . Compared with the pilot compound T3 , the inhibitory activity of H25 and H43 on PSA is increased : H25 and H43 have an inhibitory activity on PSA of 100.00 % and 87.90 % , respectively , and 60.71 % and 96.79 % respectively for the lead compound T3 and the positive control .
The results suggest that these compounds may play a role in inhibiting PC - 3 growth in androgen - independent prostate cancer cells . These results suggest that these compounds may play a role in AR - dependent and AR non - dependent dual pathways . The results suggest that these compounds may play an important role in AR - dependent and AR independent pathways . The results suggest that these compounds can be used to synthesize a series of hydantoin derivatives based on the crystal structure of ARLBD and the interaction pattern of the existing AR antagonists with ARLBD .
Based on the virtual screening strategy based on pharmacophore and molecular docking , the new leader compound T3 was found and the structure modification of T3 was used to synthesize the pyrazole - 5 - ketone derivative and pyrazole - 5 - carboxamide derivatives .
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R91;R914.5;R96
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本文編號(hào)：2043561